This invention is related to the field of treatment of cancer, other neoplastic disorders, and other conditions in vertebrates in which killing a specific group of cells is useful.
Most if not all cells of metazoan animals carry the machinery to commit suicide in a regular manner in response to suitable stimulus. This process is called programmed cell death, cell suicide, or apoptosis. Apoptosis is being extensively studied in mammals and other vertebrates, as well as in the worm Caenorhabditis elegans and the fly Drosophila melanogaster (reviews: Ellis et al., 1991; Steller, 1995). In vertebrate cells the process of apoptosis, which was previously termed xe2x80x9cshrinkage necrosis,xe2x80x9d involves a regular sequence of events, including membrane blebbing, cell shrinkage, pycnosis of nuclei with margination of chromatin, and usually cleavage of DNA into nucleosome-sized fragments (Wyllie et al., 1980).
Apoptosis is an essential part of embryonic development and of the maintenance of an adult animal. In mammals, for example, during development apoptosis plays a major role in the development of the nervous system (more than 50% of the neural cells that arise during embryogenesis undergo apoptosis), in the elimination of lymphocytes that produce antibodies which recognize self, in xe2x80x9ccarvingxe2x80x9d features such as the digits of the hand, and so forth. Throughout life, orderly apoptosis is used to eliminate damaged or unwanted cells without inducing an inflammatory reaction. Blood cells, cells of the immune system, and cells of most if not all tissues normally are eliminated by the apoptotic mechanism.
Failures of apoptosis produce or contribute to severe diseases, including autoimmune diseases and some cancers. It has been argued that one of the major causes of the development and progression of many cancers is a reduction of the occurrence of apoptosis (Wyllie, 1985; Fisher, 1994; Hickman et al., 1994, Martin and Green, 1995; Thompson, 1995).
A wide variety of signals induce apoptosis in suitable target cells (Gerschenson and Rothello, 1992; Thompson, 1995). Radiation and many valuable chemotherapeutic agents, such as cisplatin and other platinum compounds, induce apoptosis (e.g., Eastman, 1990; Hickman, 1992; Chu, 1994a). These agents affect many cell types. Specialized cell types are dependent on specific growth factors (e.g., nerve growth factor for certain neuronal cells, interleukin-2 for certain lymphocytes) and undergo apoptosis if the required factors are unavailable. Other cell types have receptors for specific agents that can induce apoptosis in these cell types (e.g., glucocorticoid for thymocytes, tumor necrosis factor in suitable target cells) (e.g., Rubin et al., 1988).
The mechanism of apoptosis is just beginning to be understood. Some have suggested that all cells are poised to die, and that they are kept alive by constant xe2x80x9csurvival signalsxe2x80x9d that keep the suicide machinery inactive (Raff, 1992). It is clear that many if not all vertebrate cells contain preformed machinery for apoptosis, since there are many examples of cells that undergo apoptosis even without synthesis of new proteins (Waring, 1990). There also are cases in which protein synthesis is required (reviewed by Cohen, 1993).
Several elements that appear to be part of the apoptotic machinery have been identified and are receiving much attention. Two that should be mentioned are bcl-2 and its family members and p53. Exactly how these are related to the apoptotic machinery is still being defined.
Expression of oncogene bcl-2 in cells markedly delays or blocks induction of apoptosis by many agents, including some that are valuable in chemotherapy of tumors, such as cisplatin (Reed, 1994; Korsmeyer, 1995; Thompson, 1995). There are a few cases in which induction of apoptosis is unaffected by expression of bcl-2 (e.g., Sentman et al., 1991; Vaux et al., 1992). High-level expression of bcl-2 is common in tumors, including breast carcinomas, small cell lung cancer, androgen-independent prostate cancer, and neuroblastoma (Hickman et al., 1994). In some cases expression of bcl-2 is correlated with a poor prognosis for therapy (Reed, 1994).
Functional tumor suppressor gene product p53 is required for induction of cell death by irradiation and many chemotherapeutic agents (Lowe et al., 1993), as well as by oxygen deficiency at the center of solid tumors (Graeber et al., 1996). On the other hand, the normal development of transgenic animals nullizygous for the p53 gene indicates that p53 is not required for the extensive apoptosis that occurs during development (Donehower et al., 1992). Other cases of p53-independent apoptosis have been described (White, 1993; Zhuang et al., 1995). Many established lines of cells in culture have lost p53 function. In tumors in vivo, loss of p53 function is common, and this loss is correlated with tumor aggressiveness and indicates a poor prognosis for treatment by standard protocols of chemotherapy and radiation (Fisher, 1994; Hartmann et al., 1997).
As an example, the roles of p53 loss and bcl-2 expression in the development and progression of colon carcinomas have been described and analyzed (Hickman et al., 1994; Sinicrope et al., 1996).
The present invention is based on our discovery that apoptotic cell death can be induced in diverse cell types by creating a deficiency in the natural vitamin, thiamin. The invention provides a method for inducing death in selected cells in vivo by using localized delivery of thiamin-depleting compounds to reduce the thiamin in these cells below a critical level. This method, localized apoptosis induced by depletion of thiamin (LAIDT), is applicable to therapy of cancer and to elimination of other targetable cells. Furthermore, the method allows rapid and convenient reversal of the effects of the deficiency at any time such reversal is desired, simply by the administration of replacement thiamin.
This method allows the selective killing of a group of cells, for example a tumor mass, by localizing the deficiency of thiamin to the desired cell group. As described below, both the thiamin depletion and the targeting can be accomplished in a variety of different ways. Typically however, the method involves the delivery of a thiamin-depleting agent or a nucleic acid sequence encoding a thiamin-depleting agent to the desired cell group. The creation of the thiamin deficiency, which results from the delivery of the thiamin-depleting agent, leads to programmed cell death, or apoptosis. This method is broadly applicable to use with cells of vertebrate organisms, which cannot produce their own thiamin and so rely on exogenously provided, i.e., dietary, thiamin to provide the cellular requirements. In particular, the method can be utilized in vivo in a vertebrate organism, for example a human.
This invention utilizes a novel paradigm for cancer theraby, in addition to those currently commonly used or tested (e.g., radiation, chemotherapy, immunotherapy, gene therapy, antiangiongenesis therapy). In this paradigm, selective starvation of cancer cells for a particular required nutrient whose absence induces apoptosis, in this description the essential vitamin thiamin, leads to death of the cancer cells.
Thus in a first aspect, the invention provides a method for inducing apoptosis of a selected group of vertebrate cells in vivo by sufficiently reducing the level of thiamin in cells of the group. For example, the cells may be neoplastic cells, e.g., cancer cells.
The term xe2x80x9capoptosisxe2x80x9d refers to the process of programmed cell death, with its accompanying cellular morphological changes and loss of cell viability. This does not mean however that all methods of inducing apoptosis or the mechanisms of cell death associated with different induction methods are the same.
In the context of this method, the term xe2x80x9cinducingxe2x80x9d means a direct or indirect causal relationship. Thus, the presence and/or maintenance of a particular condition causes or leads to the induced result.
The term xe2x80x9cvertebrate organismxe2x80x9d, as is commonly understood, refers to an animal which has a spinal column, such as birds and mammals, and specifically includes humans.
The term xe2x80x9cgroup of vertebrate cellsxe2x80x9d refers to a subset of the cells of a vertebrate organism, and thus includes cells of a human. Such a subset may, for example, be a particular tissue or localized portion of a tissue, an organ or portion of an organ, or a solid tumor. The term may also refer to cells of a particular type, for example, dispersed tumor cells. The term xe2x80x9cselectedxe2x80x9d indicates that the group of cells in which apoptosis was to be induced was chosen prior to reduction of the level of thiamin.
The term xe2x80x9cin vivoxe2x80x9d indicates that the thiamin depletion portion of the method is carried out on cells within a living organism. However, some steps or portions of steps of the described methods may be performed outside the organism. An example could be the preparation of compositions containing thiamin-depleting agents.
The phrase xe2x80x9creducing the level of thiaminxe2x80x9d means that the average concentration of thiarnin in a group of cells is reduced. xe2x80x9cReducing the level of thiaminxe2x80x9d further means that the average cellular concentration of thiamin in the affected group of cells is made lower than it would have been without the initiation of the method of this invention, with conditions otherwise being the same.
The term xe2x80x9csufficientlyxe2x80x9d indicates that the thiamin is reduced to a level at least low enough to result in the induction of apoptosis. Thus, the term indicates a functional requirement for the level of reduction. In general, this sufficient reduction can be determined empirically by standard methodology.
In general, the reduced level of thiamin for a group of cells should be maintained throughout a latent period for apoptosis of the group of cells to occur. Therefore in preferred embodiments, the reduced level of thiamin is maintained for as long as is necessary to obtain a therapeutic effect, generally by achieving death of the target cells. As indicated by in vitro results, this is likely to be at least about six days, and probably longer before treated cells, preferably all treated cells, in a population are killed. Therefore, in preferred embodiments, a reduced level of thiarnin is maintained for about 3-100 days, more preferably about 3-60 days, and still more preferably about 3-45 days. However, in some applications sufficient reduction of the level of thiamin will be achieved within other, preferably shorter times, so in other preferred embodiments, a reduced level of thiamin is maintained for about 4-30 days, more preferably about 4-20 days, 6-20 days, 10-45 days, or 10-30 days.
For the methods of this invention, a xe2x80x9ctherapeutic effectxe2x80x9d refers to an effect on a treated organism in which at least a portion of the targeted cells are killed. For the treatment of a disease or condition, a therapeutic effect preferably also results in at least a temporary alleviation of at least one symptom or reduces the severity of the disease or condition. Thus, therapeutic effects can include both temporary or transient effects and permanent effects, which can include a cure.
In some circumstances, it will be easier to maintain a sufficiently low level of thiamin in the selected group of cells if the cells of the organism as a whole are partially depleted of thiamin. In this way, the additional localized thiamin depletion that is needed is lessened. Thus in preferred embodiments, the method also involves partially depleting the organism of thiamin, preferably by providing a thiamin restricted diet to the organism. In this way the amount of available thiamin is reduced.
The term xe2x80x9cthiamin restricted dietxe2x80x9d means that the total dietary thiamin intake of the organism is sufficiently reduced over a period of time so that the total cellular thiamin content of the organism is reduced below the level which would be present with a normal dietary intake of thiamin.
In this context, xe2x80x9cavailable thiaminxe2x80x9d refers to thiamin which is in a form and location such that it can be taken up by a cell or group of cells and utilized within the cell or cells.
In preferred embodiments, the level of thiamin in the selected group of cells is reduced by administering at least one thiamin-depleting agent.
A xe2x80x9cthiamin-depleting agentxe2x80x9d is a compound which inactivates or inhibits the cellular utilization of thiamin under physiological conditions. Such an inactivation can result from various mechanisms, including, for example, cleavage, chemical modification, and sequestering of thiamin molecules. Inactivation can also occur either extracellularly or intracellularly. Inhibiting the cellular utilization of thiamin can also take place through various mechanisms, including, for example, inhibition of uptake, competitive binding, inhibition of an enzyme which has thiamin as a substrate or co-enzyme, and inhibition of an enzyme which is necessary for the cellular utilization of thiamin, but which does not directly have thiamin as a substrate or co-enzyme.
The thiamin-depleting agent of this invention can be of various types including polypeptides, peptides, chemically modified peptides or polypeptides, and various classes of peptidomimetic and other synthetic compounds, including, e.g., engineered ribozymes. In some embodiments, the agent is a chemical analogue of thiamin. Also in some embodiments, the agent is a small molecule.
The term xe2x80x9csmall moleculexe2x80x9d indicates that the molecule has a molecular weight of less than about 5,000 Daltons, preferably less than about 3,000 Daltons, more preferably less than about 2,000 Daltons, still more preferably less than about 1,000 Daltons, and most preferably less than about 600 Daltons.
A variety of different types of thiamin-depleting agents can be utilized, thus in preferred embodiments the thiamin-depleting agent is a thiamin-cleaving compound, such as a thiaminase or thiaminase derivative, or a thiamin binding compound, or a thiamin antagonist such as a thiamin antimetabolite.
In reference to thiamin-depleting agents, a xe2x80x9csynthetic compoundxe2x80x9d is a compound which has a chemical structure different from a naturally occurring compound such as thiamin, thiaminase or a naturally-occurring thiamin-binding compound or other naturally occurring protein or polypeptide having thiamin-depleting activity. In the case of synthetic polypeptide agents, they may be prepared by expression from a nucleic acid sequence, but the sequence of the synthetic polypeptide differs from the sequence of a naturally occurring thiamin-depleting agent. Such a synthetic agent may include, for example, the catalytically active portion of a naturally occurring thiaminase. It may also include a compound prepared, at least in part, by chemical synthesis methods, such as a peptidomimetic compound. A xe2x80x9cfully syntheticxe2x80x9d compound is a synthetic compound which is prepared, at least in part, by chemical synthetic methods rather than synthesis by expression from a nucleic acid sequence encoding a thiamin-depleting agent. In the case of fully synthetic polypeptide agents, the fully synthetic compound does not include an amino acid sequence which has an identical amino acid sequence as a portion at least 10 amino acids in length of a naturally occurring thiamin-depleting agent which would retain the thiamin-depleting activity, such as a catalytically active portion of a naturally occurring thiaminase. Thus, synthetic compounds include sythetic thiaminases.
Also in preferred embodiments, a polypeptide thiamin-depleting agent can be expressed from a recombinant gene or nucleic acid sequence, in which case both the nucleic acid sequence and the encoded thiamin-depleting agent can be regarded as a thiamin-depleting agent. This includes for example, expression of a thiaminase or other thiamin-depleting agent from such a nucleic acid sequence in a vertebrate organism. Preferably, for such gene therapy, the sequence encoding the thiamin-depleting agent is on a vector, preferably an expression vector, and most preferably a eukaryotic expression vector. The vector can, for example, be a retroviral vector or a plasmid vector In preferred embodiments, the methods involving nucleic acid sequences also utilize other components which are preferably associated with the nucleic acid sequence, for example, those described below for methods for delivering a nucleic acid sequence encoding a thiamin-depleting agent to vertebrate cells. In addition, the nucleic acid sequence can be provided in a composition which includes a protective component, for example, a liposome or a biodegradable nanoparticle.
In connection with delivery of thiamin-depleting agents or nucleic acid sequences encoding thiamin-depleting agents, the term xe2x80x9cprotective componentxe2x80x9d refers to a compound or preferably a physical structure which protects the agent or nucleic acid from degradation or inactivation in vivo in a vertebrate animal. Examples include liposomes, lipid/nucleic acid complexes, biodegradeable nanoparticles or nanospheres, and nucleic acid binding compounds which coat or surround nucleic acid molecules such as DNA.
In connection with nucleic acid sequences, use of the term xe2x80x9crecombinantxe2x80x9d indicates that the sequence has been transferred into or recombined into a different nucleic acid molecule. Thus, for example, a sequence encoding a thiaminase can be transferred into the nucleic acid molecule of an expression vector such as a plasmid, and is then a recombinant sequence.
A xe2x80x9cvectorxe2x80x9d refers to a structure consisting of or including a nucleic acid molecule which is suitable for transferring genetic material into a cell. Typically a selected nucleic acid sequence is inserted into the nucleic acid molecule of the vector. Examples include plasmid and viral vectors.
An xe2x80x9cexpression vectorxe2x80x9d is a vector constructed and adapted to allow expression of an inserted nucleic acid coding sequence in a cell. Thus, the vector includes nucleic acid sequences which allow initiation of transcription in an appropriate location with respect to the coding sequence. Expression vectors can be adapted for expression in prokaryotic or eukaryotic cells, thus, a xe2x80x9ceukaryotic expression vectorxe2x80x9d is constructed to allow expression of a coding sequence in a eukaryotic cell.
The term xe2x80x9cthiamin-cleaving compoundxe2x80x9d refers to a compound which is able to interact with the thiamin molecule in solution under approximately physiological conditions and cause cleavage of the thiamin molecule into at least two discrete portions. The thiamin-cleaving compound can, for example, interact with the thiamin molecule in an enzymatic interaction or can directly react with the thiamin.
Thiaminases, as found in scattered groups of organisms, are a useful natural starting point to obtain thiamin-cleaving compounds to use to induce apoptosis by reducing the level of thiamin in the environment of selected cells. In addition to the naturally occurring thiaminases, a variety of modifications and derivatives can be constructed which can have advantageous properties for use as therapeutic compounds. In general such changes are directed to making a compound which is more stable in vivo and/or which is more readily administered. A useful approach for making such changes is to trim down a naturally occurring thiaminase to identify a functional core or reduced length sequence which retains thiamin-cleaving activity in order to minimize the probability or rate of destruction of the molecule in the body of a treated animal. Such size reduction can also reduce the antigenic potential of the molecule and can aid in penetration of the molecule into in vivo sites, such as the interior of a solid tumor. Alternatively or in addition, amino acid substitutions or chemical modifications can be incorporated, which can reduce the rate of in vivo destruction. Thus, the methods of the present invention encompass the use of naturally occurring thiaminases, modified thiaminases, and thiaminase derivatives.
The natural thiaminases can also be used to design synthetic or artificial thiamin-cleaving compounds, which can be regarded as synthetic or artificial thiaminases. Based on comparison of a variety of thiaminases and the structural requirements for thiamin-cleaving function, such as structural studies of the active sites, synthetic thiaminases can be constructed. For example, peptides of small, or minimal size, can be constructed having thiamin-cleaving activity using this approach. Preferably such peptides will have less than about 200 amino acid residues, more preferably less than about 100, still more preferably less than about 75, and most preferably less than about 50 or even 20 amino acid residues. Such peptides can be prepared by chemical synthesis or by expression from nucleic acid sequences encoding the amino acid sequence. Chemical modifications of one or more amino acids can also be incorporated.
The term xe2x80x9csynthetic thiaminasexe2x80x9d refers to a thiamin-cleaving compound which preferably is structurally based on the catalytic site of a natural or reduced size thiaminase, but which has a different chemical structure than the natural active site. Thus, the synthetic thiaminase can have a different amino acid sequence, or can include non-natural amino acids, or can include non-amino acid structures, or can have a completely non-amino acid structure. Alternatively, a synthetic thiaminase may have a structure unrelated to the catalytic site of a natural thiaminase, but the molecule has a thiamin-cleaving activity. A xe2x80x9csynthetic thiamin-binding compoundxe2x80x9d is similarly related to naturally occurring or reduced sized polypeptide thiamin-binding compounds.
The structural and functional analysis can also be used to construct artificial thiaminases, which incorporate non-natural amino acids and/or other chemical structures, but which also specifically cleave thiamin. Such compounds can be termed peptidomimetics, as they mimic the function and structure of a peptide or polypeptide. While such compounds can be obtained in many different ways, one useful approach is to use a combinatorial approach to synthesize libraries of potential thiamin-cleaving compounds based on the active sites of thiaminases. These libraries can then be tested for activity against thiamin and for the ability to induce apoptosis. The incorporation into the molecule of elements which are not natural amino acids allows particular properties of the compound to be enhanced. Such properties can include in vivo stability against degradation (chemical or physical), improved penetration of cells or tissues, and decreased antigenicity.
A similar approach is also applicable to other naturally occurring polypeptide thiamin-depleting agents, for example, to naturally occurring polypeptide thiamin-binding compounds. As with the thiaminases, the naturally occurring thiamin-binding compounds can be trimmed down to identify reduced or minimal size thiamin-binding portions, which can be further modified with the incorporation of non-natural amino acids or other chemical modifications. Also as above, identification of the structure of the binding region allows the design and construction of synthetic peptides or peptidomimetic compounds based on that structure.
As used herein, the term xe2x80x9cthiaminasexe2x80x9d refers to a polypeptide molecule which is a naturally occurring thiamin-cleaving enzyme, or which is a modification of such an enzyme. Examples of naturally occurring thiaminases are described below in the Detailed Description. A modified thiaminase is based on a naturally occurring thiaminase but contains one or more modifications to the amino acid sequence of the polypeptide. Such changes can include the addition or deletion of one or more amino acids, the substitution of one or more amino acids with other amino acids or combinations of such changes, and chemical modification of one or more amino acid residues. Changes of these types can, for example, be used to provide one or more functional advantages over a corresponding natural thiaminase, including, for example, increased stability to enzymatic or chemical degradation, decreased antigenicity, and improved penetration of the modified thiaminase among target cells.
For amino acid additions, in preferred embodiments the addition is a terminal addition providing an amino acid sequence which has a functionality different from a thiaminase, for example, a cell targeting function. For deletions, the resulting modified thiaminase retains the thiamin-cleaving activity of the natural thiaminase. In some embodiments, a modified thiaminase having a deletion preferably contains the majority of the amino acid sequence of the corresponding naturally occurring thiaminase, for example, the deletion may be less than 40%, 30%, 20%, 10%, 5%, or 2% of the naturally occurring thiaminase. Combinations of amino acid additions and deletions may also be present in the same modified thiaminase. For amino acid substitutions, the substitution does not destroy the thiamin-cleaving activity of the modified thiaminase, preferably the modified thiaminase has substantially the activity of the corresponding natural thiaminase. Such substitutions preferably are present in only a small number of sites in the modified thiaminase, for example, in less than 10%, 5%, or 1% of the sites of the naturally occurring thiaminase. In addition to the above changes, a thiaminase may also be modified by chemical modification of one or a few amino acid residues of the molecule. Preferably, but not necessarily, such modification occurs at less than 10%, 5%, or 1% of the amino acid residues. In all of the cases, the thiaminase or modified thiaminase has a thiamin-cleaving activity.
A xe2x80x9cthiaminase derivativexe2x80x9d refers to a compound which is a polypeptide which includes a sequence of amino acids similar to at least a portion of a natural thiaminase and which retains thiamin-cleaving activity, but which has one or more modifications from the natural thiaminase. One type of derivative corresponds to a portion of a natural thiaminase. This means that the derivative is based on the amino acid sequence of one or more linear parts of the natural thiaminase but less than the full molecule, and retains thiamin cleaving activity. Thus, the derivative may be a linear fragment of the thiaminase or a combination of more than one linear fragments joined together. The derivative may, for example, include at least 20%, 40%, 60% or more of the amino acid sequence of the corresponding naturally occurring thiaminase. In thiaminase derivatives, the amino acid sequence can also be modified, such as by the substitution, deletion, or insertion of one or a few amino acids or combinations of these. Derivatives can also have other modifications. For example, non-natural amino acids can be utilized and side chain modification of natural amino acids can be performed as understood by those skilled in the art. Such modifications can, for example, result in greater resistance to protease cleavage. In certain derivatives, combinations of the above changes may be utilized.
A xe2x80x9cthiamin-binding compoundxe2x80x9d refers to a compound which preferentially forms a stable association with thiamin in approximately physiological conditions. The association is sufficiently stable to effectively sequester thiamin. For compounds to be used in the methods of this invention for inducing apoptosis, the association effectively prevents a cell from utilizing the sequestered thiamin. As with the description of thiaminase above, the term xe2x80x9cthiamin-binding compoundxe2x80x9d includes modifications of the naturally occurring molecule.
The term xe2x80x9cthiamin-binding compound derivativexe2x80x9d refers to a compound which is related to a naturally occurring thiamin-binding compound in the manner indicated for thiaminases and thiaminase derivatives above.
xe2x80x9cThiamin antagonistxe2x80x9d refers to a compound which has an activity in a cell antagonistic to the cellular utilization of thiamin without altering or sequestering thiamin. Thus, the anti-thiamin activity of the compound is not primarily due to a thiamin-cleaving or thiamin-binding activity. An example of such a compound is a xe2x80x9cthiamin analoguexe2x80x9d or xe2x80x9cthiamin antimetabolitexe2x80x9d. Such an analogue has sufficient structural similarity to thiamin to compete for binding with thiamin and thereby inhibit the cellular uptake or utilization of thiamin. In most cases the activity of an analogue will be due to the binding competition, however, such an analogue may also alter a cellular molecule necessary for the cellular uptake or utilization of thiamin.
In many applications, it is beneficial for the thiamin-depleting agent to act preferentially or exclusively on the selected group of cells. Such preferential activity or selectivity can be provided by targeting the agent to the selected group of cells. Preferably the selected group of cells are cells of a tumor or vascular epithelial cells, or tumor vascular epithelial cells. Targeting can be accomplished by a number of different approaches, which include both molecular targeting approaches and physical targeting approaches and combinations of these. Therefore, in preferred embodiments, such targeting is provided. Such targeting can be provided by a variety of different mechanisms, including but not limited to localized administration of the agent, localized activation of the agent, localized expression of a nucleic acid sequence encoding the agent, and localized binding of the agent or an associated targeting molecule. A number of examples are described in the Detailed Description below.
In the context of the present methods, xe2x80x9clocalizedxe2x80x9d means that the action occurs to a greater degree in or at restricted locations, preferably to a much greater degree, rather than at other locations throughout the body of an organism. This does not mean that the action occurs at only a single location. Generally the locations are selected as having a particular property or properties, e.g., locations surrounding the site or administration of a compound, or the locations of cells which have particular surface protein.
In connection with two molecules, the term xe2x80x9cassociatedxe2x80x9d refers to a direct or indirect physical interaction such that the two molecules remain in proximity to each other or to a complex including the two molecules to a greater extent or for a longer time than non-associated molecules. Preferably the association is due to interactions such as binding, of any type, or encapsulation.
As indicated, targeting can be provided by a xe2x80x9ctargeting moleculexe2x80x9d or xe2x80x9ctargeting compoundxe2x80x9d. Such a molecule can, in certain embodiments, be free of covalent bonding with other molecules, or can be covalently linked with a thiamin-depleting agent or another component of a composition for delivery of a thiamin-depleting agent or nucleic acid encoding a thiamin-depleting agent to a cell. Thus, a xe2x80x9ctargeting moleculexe2x80x9d or xe2x80x9ctargeting compoundxe2x80x9d is a molecule which has structural characteristics which cause the molecule to preferentially locate to a limited in vivo location or type of location. Preferably and most commonly, the preferential locating is due to specific or preferential binding of the targeting molecule to a particular molecule in the organism which is exclusively or at least predominantly found in association with a particular group of cells. However, other distributions or interactions can also be used, such as tight binding to a widely distributed cellular molecule following localized administration, thus preventing diffusion or transport of the thiamin-depleting molecule to other locations.
The targeting can, for example, be provided by an antibody, antibody fragment, or a derivative of an antibody which recognizes an appropriate cellular antigen on the target cells. The targeting can also be provided by a cellular receptor ligand. A xe2x80x9ccellular receptor ligandxe2x80x9d refers to a molecule or a portion of a molecule which is recognized and bound by a cellular receptor, preferably a receptor accessible on the cell surface. Such receptors are generally proteins which are able to specifically or at least preferentially bind particular molecules or ligands. Commonly, the binding of a natural ligand is linked with a further biological response or effect. Additional targeting molecules can be obtained using phage-display or combinatorial libraries to identify peptides or peptidomimetics or other molecules which specifically or preferentially bind to particular tissues or cells. An example is the identification of peptides which preferentially bind to epithelial markers, such as tumor endothelial cell markers or tissue specific capillary cell markers.
Such targeting molecules can be used in a variety of different ways, including, for example, direct attachment or association of the targeting molecule with the thiamin-depleting compound, and attachment or association of the targeting molecule with a complex which includes a thiamin-depleting compound. Examples of such complexes include complexes using protective components such as liposomes, nanospheres or nanoparticles which are preferably biodegradable, or biocompatible gels. Such complexes can be used for delivery of many different types of compounds. In an example particularly useful for delivery of nucleic acid sequences, nucleic acid binding compounds, e.g., polylysine, spermine, or other DNA condensing compounds, can be attached to a targeting molecule and allowed to bind to the nucleic acid.
The targeting can also be provided by localized administration, meaning that the thiamin-depleting agent is introduced into the organism in a specific location which results in exposure of the targeted cells to the thiamin-depleting agent. Preferably, the location of introduction is among or around the targeted cells. An example of such localized administration is direct injection, for example, injection into and/or around a tumor. Localized administration can be utilized either alone or in conjunction with other targeting technique or techniques to enhance the localization effects. As another example, direct localized administration can be provided by inhalation or instillation of the thiamin-depleting compound agent into the lungs.
The targeting can also be provided by intravenous injection of compositions having particular selections of cationic lipids upstream of a capillary bed. An example of such a cationic lipid is the cationic lipid known as DOTMA. In using such cationic lipids, it is often beneficial to utilize a neutral co-lipid, for example, cholesterol or DOPE.
xe2x80x9cCationic lipidxe2x80x9d refers to a compound having a lipid structure as understood by those skilled in the art, which has a net positive charge in aqueous solution at physiological pH. A xe2x80x9cneutral lipidxe2x80x9d or xe2x80x9cneutral co-lipidxe2x80x9d is a lipid compound which is uncharged or very nearly uncharged in aqueous solution at physiological pH.
In another example, the targeting by localized administration can also be accomplished by intra-arterial infusion into an artery supplying a localized tumor. Generally this will utilize a surgically implanted catheter and an infusion pump.
Yet another example of targeting is the use of a thiamin-depleting agent where the agent is inactive until activated in a localized manner or the agent activates an agent which thus becomes toxic to cells. An example of such activation is a localized prodrug activation, in which an inactive prodrug is locally converted to an active drug form. Such prodrugs can be of various types, and the localized activation can, correspondingly, be accomplished in various ways. For example, as described in the Detailed Description, such prodrug activation can be accomplished using prostate specific antigen (PSA) in an example of localized activation of a thiamin-depleting agent, e.g., a thiaminase. Another example uses antibody directed enzyme prodrug therapy, in which an enzyme is attached to a targeting antibody (i.e., a targeted enzyme). The attached enzyme then activates an administered prodrug locally at the targeted cells. The prodrug is only toxic in its activated form, and thus kills cells in situ. The prodrug can be a thiamin antimetabolite, e.g., a thiamin analogue. For example, a tissue-targeted thiaminase can cleave a thiamin analogue prodrug to produce a toxic compound that kells cells. Other types of targeting molecules could also be used in this manner.
Yet another exemplary targeting method utilizes the hypoxic or anaerobic interior of solid tumors as a targeted environment. One approach uses a gene expression control element which is inducible under hypoxic conditions to control the expression of a nucleotide sequence encoding a thiamin-depleting agent, e.g., a thiaminase or derivative. Some elements of this type have been termed xe2x80x9chypoxic responsive elementsxe2x80x9d or xe2x80x9cHRExe2x80x9d, for example elements which are binding sites for the transcriptional complex, hypoxia inducible factor-1 (HIF-1). A coding sequence controlled by a hypoxia-inducible element will be expressed at a significantly higher level, e.g., preferably at least 2-fold and more preferably at least 5-fold higher in a hypoxic tissue environment than in normally oxygenated tissue. The nucleic acid encoding the thiamin-depleting agent can be delivered in a variety of different ways, but typically will be on a vector, such as a viral, e.g., retroviral, or plasmid vector. The vector can be located to the hypoxic interior of solid tumors using a number of different methods as known to those skilled in the art. Examples include direct injection into a tumor, localization using antibodies, antibody fragments, targeting proteins, peptides, or ligands which preferentially bind to proteins primarily present on tumor cells, intravenous injection upstream of a tumor, or any method which will allow the vector to penetrate into the tumor. A second approach uses anaerobic bacteria, such as Clostridium spp., to target a thiamin-depleting agent to the interior of solid tumors. The bacteria can, for example, be modified to secrete a thiamin-depleting agent, such as a thiaminase, or can express a prodrug activating enzyme, e.g., prodrug cleaving, which will activate an inactive prodrug of a thiamin-depleting compound.
The terms xe2x80x9chypoxicxe2x80x9d and xe2x80x9canaerobicxe2x80x9d refers to a lower oxygen tension or concentration which is lower than in most similar environments. Thus, a tissue, e.g., the interior of a tumor, is hypoxic if the concentration of dissolved oxygen is significantly lower than in most normal tissues of the same organism or similar organisms. Generally a hypoxic tissue region will have an oxygen partial pressure of less than about 20 mmHg, preferably less than about 10 mmHg, and most preferably less than about 5 or 3 mmHg. In contrast, normal tissue will generally have an oxygen partial pressure of greater than 20 mmHg, typically in the range of about 24-66 mmHg.
Non-pathogenic bacteria can also be used to deliver a thiamin-depleting compound such as a thiaminase by colonizing a particular part of the body (i.e., localized colonization) with an appropriate bacterium expressing the compound. For example, particular bacteria are known which colonize different parts of the intestines, the vagina, and the bladder. Other parts of the body can be protected, as needed, through measured administration of thiamin.
In connection with the various targeting techniques, those skilled in the art will recognize that such targeting can be utilized for both the administration of thiamin-depleting agents and for the administration of nucleic acid sequences encoding thiamin-depleting agents.
In a preferred embodiment, the invention provides a method of inducing apoptosis of a selected group of vertebrate cells in vivo by reducing the level of thiamin in the cells sufficiently to induce apoptosis by administering a plurality of thiamin-depleting agents to the organism. The plurality of thiamin-depleting agents may belong to a single class, such as thiaminases, or may be a combination of agents drawn from different classes or combinations of these possibilities. Thus, in preferred embodiments at least one of the thiamin-depleting agents is a thiamin-cleaving compound, such as a thiaminase or thiaminase derivative, a thiamin binding compound, or a thiamin antagonist or antimetabolite. In certain embodiments at least one of the agents is a thiamin-cleaving compound, e.g., a thiaminase, and at least one is a thiamin-binding compound; the thiamin-cleaving compound can be provided or act extracellularly and the thiamin-binding compound can be provided or act intracellularly. Similarly, in certain embodiments at least one of the agents is a thiamin-cleaving compound, e.g., a thiaminase, and at least one is a thiamin antagonist; the thiamin-cleaving compound may be provided or act extracellularly and the thiamin antagonist can be provided or act intracellularly. In a similar manner as discussed above, peptide or polypeptide agents can be encoded on a vector and are expressed from a recombinant gene in the organism. Likewise, in preferred embodiments, the plurality of thiamin-depleting agents includes a plurality of thiamin-cleaving agents, such as a plurality of thiaminases, a plurality of thiamin-binding compounds, or a plurality of thiamin antimetabolites. In preferred embodiments, a plurality of thiamin-depleting agents can be administered sequentially or can be administered concurrently. Likewise, a plurality of agents can be administered in two or more sets, in which the members of a set are administered concurrently and the individualy sets are administered sequentially. Each set contains one or more thiamin-depleting agents.
Embodiments of the method using a plurality of thiamin-depleting agents include a variety of specific choices, such as those described herein in other embodiments of this aspect for particular thiamin-depleting agents, methods and agents for localizing, targeting compounds, compounds which enhance bioavailability of an agent, compounds which enhance recombinant gene delivery and/or expression, and other selections.
In cases in which the organism reacts immunologically to a thiamin-depleting agent, sequential administration of a plurality of thiamin-depleting agents can be used to avoid excessive immune response which could interfere with the thiamin depletion. In this case, a shift is made to an immunologically unrelated agent before the development of a strong immune response to the prior agent. Thus, the shift is timed to reduce the immune response to any of the plurality of agents.
In preferred embodiments, the method uses at least one peptide or polypeptide thiamin-depleting agent. As indicated above, this can lead to an immune response to that agent. Therefore, in preferred embodiments, the method also includes administering an inactive analogue of the polypeptide, e.g., a thiaminase or thiaminase derivative, thereby inducing immunologic tolerance to the inactive and corresponding active peptides polypeptides. Preferably, the inactive analogue is not targeted to the selected group of cells.
An xe2x80x9cinactive analoguexe2x80x9d refers to a compound which does not have appreciable activity of a particular type, which is structurally similar to a corresponding compound which possesses a significant level of the particular activity. For use for inducing tolerance, an inactive analogue preferably has the same major epitopes as a corresponding active compound, and preferably has only minimal changes necessary to cause a loss of the activity.
As used herein, the term xe2x80x9ctolerancexe2x80x9d refers to immunologic tolerance, and thus indicates a reduced responsiveness of an organism""s immune system to a particular antigen. Generally immunologic tolerance involves the antigen-specific inactivation or deletion of particular B- or T-lymphocytes. As understood by those skilled in the art, such tolerance can develop in several different ways with variations in the underlying biological processes. For example, it is understood that the mode of antigen exposure is frequently an important factor in the development of tolerance.
In other preferred embodiments, the method of inducing apoptosis provides a method for treating a neoplastic disorder in a vertebrate organism. In this method the selected group of cells are cells of the neoplastic disorder. In preferred embodiments, the neoplastic disorder is a cancer and the vertebrate organism is a human. Also in preferred embodiments, cells of the cancer form a solid tumor. Other preferred embodiments of the method for treating a neoplastic disorder are as described above or otherwise described herein, including embodiments in which a plurality of thiarnin-depleting agents is used.
The term xe2x80x9cneoplastic disorderxe2x80x9d refers to a condition in a complex organism, such a vertebrate, in which there is an abnormal mass of tissue, including dispersed cells, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, the term includes neoplastic growths or tumors. Neoplastic disorders particularly include xe2x80x9ccancerxe2x80x9d or malignant tumors. A large number of different cancers are known to those skilled in the art, the cells of which can be induced to undergo apoptosis.
A xe2x80x9csolid tumorxe2x80x9d refers to a localized mass of cancer cells which form a macroscopic group of cells and which is physically distinct from the surrounding tissue. The term includes both encapsulated and nonencapsulated tumors. Thus, the boundary between the tumor mass and normal tissue is not necessarily a discrete boundary.
The method of treating a neoplastic disorder by reducing the level of thiamin in the cells of the neoplastic disorder can also be used in conjunction with other anti-neoplastic treatments. Thus, in preferred embodiments, the invention provides a method of treating a neoplastic disorder which involves reducing the level of thiamin in cells of a neoplastic disorder in order to induce apoptosis and also administering a second anti-neoplastic treatment to the organism. The second anti-neoplastic treatment can be of a variety of types, such as those commonly currently utilized, e.g., radiation and treatment with cytotoxic agents, which preferentially kill growing cells. Also in a preferred embodiment, the second anti-neoplastic treatment induces apoptosis of growing cells.
In preferred embodiments the order and timing of the thiamin depletion and the second antineoplastic treatment include a number of different regimes, for example concurrent administration of the two treatments, thiamin depletion first followed by the second antineoplastic treatment, and administration of the second antineoplastic treatment first followed by the thiamin-depleting treatment. Other preferred embodiments are as described above for localized thiamin depletion.
Similarly, in a related aspect the invention provides a method of killing a selected group of verterbrate cells in vivo by reducing the level of thiamin in the targeted cells in localized thiamin deficiency induced apoptosis (LAIDT) and, in conjunction, administering to the animal containing those cells an accessory treatment which enhances the effectiveness of the thiamin reduction. A variety of different accessory methods can be used to enhance the effectiveness of the thiamin deficiency induced apoptosis. In methods involving the use of an accessory treatment, the thiamin deficiency induced apoptosis is targeted to the selected cells, for example, by targeting methods as described herein. In addition, the accessory treatment may also be targeted unless otherwise indicated. Again, a variety of different targeting methods may be used as appropriate for the type of composition to be delivered. Such methods include, for example, the targeting methods described herein in connection with creation of a localized thiamin deficiency. However, persons familiar with the delivery of the therapeutic compositions will recognize that a variety of other methods may also be used and will readily understand the selection of appropriate methods.
In a preferred embodiment, the accessory treatment involves the elevation of a carbohydrate, preferably glucose, in the selected cells. While the carbohydrate level elevation may be localized or targeted to the selected cells, generally such localization is not necessary. Such a carbohydrate level elevation can be accomplished readily, for example, using an intravenous solution containing a particular carbohydrate, e.g., glucose. Alternatively, a dietary supplement containing the desired carbohydrate can be provided.
In other preferred embodiments, the accessory treatment involves inhibiting the formation or the function of tumor vasculature or inhibiting the ability of tumors to invade surrounding tissue. Thus, such accessory methods can be, for example, antiangiogenesis methods, methods which induce inflammation in tumor neovasculature, or methods which inhibit the modification of intracellular matrix material in tumor formation. A large number of angiogenesis inhibitors are known, including those specifically mentioned in the detailed description below and active analogs and derivatives of those compounds. As indicated, examples of such inhibitors include small molecules, antibodies, other polypeptides, and nucleic acid molecules. In particular, such nucleic acid molecules include ribozymes and other catalytic nucleic acid molecules such as those obtained by in vitro combinatorial selection techniques as well as other selection and evolution methods. In general, such nucleic acid inhibitors and antibody inhibitors are targeted to a gene product which is needed for tumor angiogenesis.
In this context, the phrase xe2x80x9cneeded for tumor angiogenesisxe2x80x9d means that elimination of the activity, including elimination of expression, of the gene product at least slows the process of tumor angiogenesis and preferably stops tumor angiogenesis. Preferably, activity of the gene product does not need to be completely eliminated but merely reduced in order to inhibit tumor angiogenesis.
With respect to the present invention, the term xe2x80x9ccatalytic nucleic acid moleculexe2x80x9d refers to a molecule which contains a plurality of nucleotides and/or nucleotide analogs and which can act to catalyze a reaction on another molecule, usually another nucleic acid molecule. Preferably a majority of the catalytic nucleic acid molecule is composed of nucleotides or nucleotide analogs. In most cases the molecule catalyzes a cleavage reaction. The term includes xe2x80x9cribozymesxe2x80x9d. This term refers to catalytic nucleic acid molecules which are based on the structure of naturally-occurring self-cleaving RNA sequences, and which retain the general structural motif of the natural sequence. As indicated below, ribozymes and other catalytic nucleic acid molecules can contain a variety of substitutions of ribonucleotides with deoxyribonucleotides, nucleotide analogs, and non-nucleotidic linkers or terminal moieties so long as catalytic activity is retained.
Also as indicated in preferred embodiments, the accessory treatment involves inhibiting the expansion of a tumor in a surrounding tissue. In a particular embodiment the accessory method involves inhibiting the action of matrix modifying enzymes, for example, matrix metalloproteinases (MMPs).
In yet another preferred embodiment, as indicated, the method involves inhibiting the function of tumor neovasculature. This can be accomplished, for example, by inducing inflammation in the tumor vasculature, thereby inhibiting the transport of nutrients to and waste products from the tumor cells. It is expected that this results in one or more of: slowing tumor growth, inducing quiescence of actively growing tumor cells, and killing tumor cells. Any such effects will result in enhancing the effectiveness of localized thiamin deficiency induced apoptosis.
The terms xe2x80x9ctumor vasculaturexe2x80x9d or xe2x80x9ctumor neovasculaturexe2x80x9d refer to the blood vessels which develop to provide a blood supply to a tumor, as distinguished from vasculature which primarily functions to supply normal tissue.
In other embodiments, a method involves modulating the level of activity of an apoptosis related protein. As described below, such apoptosis related proteins include both apoptosis suppressing proteins and apoptosis enhancing proteins. Thus, such modulation can be accomplished, for example, by increasing the level of activity of one or more apoptosis enhancing proteins and/or by decreasing the level of activity of one or more apoptosis suppressing proteins. The up regulation can be accomplished, for example, by administration of a compound which either increases the production of such an apoptosis enhancing protein or which increases the sensitivity of cells to the presence of such a protein. Included in the methods for increasing the production of an apoptosis enhancing protein are gene therapy methods providing expression of a recombinant coding sequence encoding the protein or a biologically active fragment or derivative of the protein. On the other hand, inhibition of apoptosis supressing proteins can utilize a variety of different types of inhibitors, including, for example, small molecules, antibodies, and nucleic acid inhibitors, including ribozymes and other catalytic nucleic acid molecules and antisense and triple helix inhibitors. Preferably, the apoptosis related protein is a secreted protein such as those which have been identified in the literature as secreted apoptosis related proteins (SARPs).
The term xe2x80x9capoptosis related proteinxe2x80x9d refers to a polypeptide which is involved in the process of apoptosis or a polypeptide whose presence or absence affects the sensitivity of cells to signals leading to apoptosis. In preferred embodiments, the apoptosis related protein is a receptor, a receptor component, or a receptor ligand.
In yet another preferred embodiment, the accessory treatment involves the administration of a prodrug. Preferably, activation of the prodrug is targeted to the selected group of cells. Generally, such prodrugs are activated by chemical modification or cleavage of the prodrug molecule yielding an active molecule. As understood, a variety of different activator enzyme prodrug combinations can be utilized. In particular, a thiamin cleaving compound, for example, a thiaminase or thiaminase derivative, can be used to cleave a prodrug where the cleavage results in the production of a molecule toxic to surrounding cells. In most cases, such a molecule would have structural similarities to thiamin.
In another preferred embodiment, the accessory treatment involves the administration of a second apoptosis inducing treatment. Preferably, but not necessarily, the apoptosis induction pathway for the second method involves at least a portion of the pathway involved in thiamin deficiency induced apoptosis. Such a second apoptosis inducing method can, for example, involve the administration of a compound which induces apoptosis.
In yet other embodiments, as indicated above, the accessory method can involve the creation of a generalized thiamin deficiency. Alternatively, particularly in connection with actively growing cancer cells, the generalized thiamin deficiency can be utilized alone to induce apoptosis of the rapidly growing cells of a tumor which should be more sensitive to the thiamin deficiency. It is expected that such actively growing cells would deplete the intracellular thiamin more rapidly than normal cells, and would therefore enter apoptosis sooner. After apoptosis of at least some of the actively growing cells, had occurred, thiamin can then be administered to prevent death of normal cells. Such generalized thiamin deficiency can also be used in conjunction with other treatment methods, for example, other methods as described herein for use as accessory methods in conjunction with localized thiamin deficiency.
Also, as indicated above, the accessory treatment includes the administration of a second antineoplastic treatment, for example, the administration of an antineoplastic agent. Such antineoplastic agents include, for example, conventional antineoplastic, e.g., anticancer agents that also include other methods for the inhibition and/or killing of neoplastic cells.
In another aspect, the invention features the use of a thiamin-depleting agent in the preparation of a medicament effective for the treatment of a disease or disorder in a vertebrate, such as a mammal, in which the elimination of a group of cells provides a therapeutic benefit. Such diseases or conditions include, for example, neoplastic disorders, particularly including cancers. In preferred embodiments, the medicament contains other components as described herein for pharmaceutical compositions and for compositions in the methods of inducing apoptosis, treating cancer, killing a selected group of cells, and compositions in other aspects described herein.
A xe2x80x9ctherapeutic benefitxe2x80x9d refers to a reduction in the number of cells of the condition being treated, or an improvement in at least one symptom, or an improvement in the condition. An improvement in condition can include a cure.
As indicated above, certain thiamin-depleting agents can be delivered by the delivery of a nucleic acid sequence encoding the agent, such as a thiaminase or a thiaminase derivative, and expressing the agent from that nucleic acid sequence. Therefore, in another aspect, the invention provides a method for delivering a nucleic acid sequence encoding a thiaminase or derivative thereof to vertebrate cells in vivo by delivering a vector which includes a nucleic acid sequence encoding the thiaminase or thiaminase derivative to the cells.
As recognized by those skilled in the art, it is often advantageous for delivery of nucleic acid coding sequences to vertebrate cells in vivo to prepare the nucleic acid in a composition, complex, or formulation which can be selected to have a variety of different components, depending on the particular application. For example, the nucleic acid or vector bearing the coding sequence can be prepared associated with a cationic lipid. Examples of such cationic lipids include, for example, DOTMA, DOTAP, DDAB, DOSBA, CTAB, DC-chol, and DMRIE. In these formulations it can also be advantageous to incorporate an additional lipid, generally an essentially neutral co-lipid, such as DOPE or cholesterol. These compounds are known to those skilled in the art, and are identified, for example, in Felgner et al., Cationic Lipids for Intracellular Delivery of Biologically Active Molecules, U.S. Pat. No. 5,264,618, issued Nov. 23, 1993, and in Gao and Huang, 1995, Gene Therapy 2:710-722. The structures of a variety of useful lipids are shown in the Gao and Huang reference. DOPE is the abbreviation for dioleoylphosphatidylethanolamine. DOSPA is 2,3-dioleoyloxy-N-(2(sperminecarboxyamido)-ethyl)-N,N-dimethyl-1-propanaminium trifloroacetate. DC-chol is 3-xcex2(N-(Nxe2x80x2,Nxe2x80x2-dimethylaminoethane)carbamoyl) cholesterol. DOTMA is N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride. DOTAP is 1,2-dioleoyloxy-3-(trimethyl ammonia) propane. The above references are hereby incorporated by reference in their entireties, and show both appropriate lipid compounds and methods of using those lipids for delivery of biologically active molecules including polypeptides and nucleic acid sequences.
A further example of the use of lipid DNA complexes is provided by Brigham, Method of In Vivo Delivery of Functioning Foreign Genes, International Application PCT/US90/05993 which is hereby incorporated by reference. The Brigham reference describes the use of DNA liposome preparations which produced transient expression in lung tissue of the sequence driven by a metalathianine promoter following intravenous injection. The results indicated that such lipid DNA formulations could be used to transfect cells surrounding the first capillary bed downstream from the point of injection.
In preferred embodiments, the DNA can also be associated with a DNA-binding compound, a cell-permeabilizing lytic agent, and a targeting compound, either individually or in combination. Examples of such components and the use of such complexes is described, for example, in Smith et al., Nucleic Acid Transporters for Delivery of Nucleic Acids into a Cell, International Application PCT/US96/05679, International Publication No. WO 96/40958 and in Curiel et al., Composition for Introducing Nucleic Acid Complexes and to Higher Eucaryotic Cells, U.S. Pat. No. 5,547,932, issued Aug. 20, 1996. These references are hereby incorporated by reference.
xe2x80x9cDNA binding compoundsxe2x80x9d refer to compounds as understood by those skilled in the art which are able to form strong associations with DNA under approximately physiologic conditions. The interaction between the binding compound and the DNA can involve one or more of a variety of different physical interactions, including but not limited to charge-charge interactions, H-bonding interactions, van der Waals interactions, and hydrophobic interactions. Examples include polylysine and spermine among others. Preferably the binding compound binds significantly more tightly to dsDNA than to RNA. xe2x80x9cDNA condensing compoundsxe2x80x9d are binding compounds which are able to collapse dsDNA so that the DNA molecule occupies a significantly smaller solution volume.
xe2x80x9cCell permeabilizing lytic agentsxe2x80x9d are compounds, e.g., certain peptide sequences, which enhance at least the local permeability of a cell to passage of other molecules across the cell membrane. Thus, a compound which is associated with or which is also present at a cell membrane with the agent is more likely to enter the cell than in the absence of the lytic agent. As described below, it is known that certain types of peptide sequences act as lytic agents and enhance the entry of associated compounds into a cell. Such associated compounds can be of various types, including, for example, DNA polynucleotides.
In addition to targeting compounds or ligands, targeting can also be provided in the case of nucleic acid sequence delivery by localized expression. Among other methods, such localized expression can be provided by using gene switch methods as described in the literature, including gene switches using an inducible promoter, such as one induced by a steroid. Thus, localized administration of the appropriate switching agent will result in induction of expression only, or at least primarily in the close vicinity of the administered inducing agent. A variety of such inducible promoters are known in the art and can be utilized for such gene switches.
Also in preferred embodiments, the DNA is associated with a compound which enhances the bioavailability of the DNA. The phrase xe2x80x9cenhances the bioavailabilityxe2x80x9d means that the compound increases the total availability of an associated molecule for participation in a particular response or action. Thus, for example, the enhancing compound can increase the time before degradation or the time at a location of an associated molecule. A number of such compounds have been described, for example, in Rolland et al., Formulated Nucleic Acid Compositions and Methods of Administering the Same for Gene Therapy, International Application PCT/US96/17038, International Publication WO 96/21470. Such compounds include, for example, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, propylene glycol, and chitosan. As indicated, such compounds can be utilized in the delivery of nucleic acid molecules, but can also be used in the delivery of other agents, such as the thiamin-depleting agents described herein.
As indicated above, targeting can be provided by components such as antibodies and receptor ligands. The encoded thiamin-depleting agent can be any of a variety of polypeptide compounds, such as polypeptide thiamin- cleaving compounds, including thiaminases and thiaminase derivatives, and polypeptide thiamin-binding compounds or derivatives.
With the exception of the thiaminase of Bacillus thiaminolyticus, the majority of the naturally occurring thiaminases are encoded by nucleic acid sequences which have not previously been isolated. Therefore, the invention also provides a purified, enriched, or isolated nucleic acid sequence encoding a thiaminase or a derivative of a thiaminase which is from such another source or which differs from a full-length naturally occurring thiaminase from Bacillus thiaminolyticus. However, it should be clear that nucleic acid sequences encoding full-length Bacillus thiaminolyticus thiaminase can also be utilized for the present invention, directly or obtaining derivatives or other related thiamin-cleaving compounds.
In a preferred embodiment, the thiaminase is from a Naegleria species, such as Naegleria gruberi. In another preferred embodiment, the thiaminase is from a fern or other pteridophyte, such as the fern bracken (Pteridium aquilinum) or the fern nardoo (Marsilea drummondii). In still another, the thiaminase is from a fish, preferably of the family Cyprinidae, such as carp.
In the case of nucleic acid sequences encoding thiaminase derivatives, it is often advantageous for the encoded amino acid sequence to be shorter than a full length naturally occurring thiaminase. Therefore, in preferred embodiments, the nucleic acid sequence encodes a modified thiaminase or thiaminase derivative containing about 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less or the amino acid sequence of the corresponding natural thiaminase. Thus, for example, the nucleic acid sequence can encode a derivative having about 400 or fewer, 200 or fewer, 100 or fewer, or 50 or fewer amino acids. Similarly, the nucleic acid sequence can encode a polypeptide thiamin-binding compound or derivative.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant a polymer of nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated or synthesized (e.g., cDNA) nucleic acids of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide sequence present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it and thus is meant to be distinguished from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold greater, more preferably  greater than 100-fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5- to 10-fold, more preferably at least 100- to 1000-fold, or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector. This term distinguishes the sequence from naturally occurring enrichment events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of MRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml, more preferably at least 100- or 1000-fold greater). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least three orders of magnitude, and more preferably four or five orders of magnitude is expressly contemplated. The term is also chosen to distinguish clones already in existence which may encode a thiaminase or portion of a thiaminase but which have not been isolated from other clones in a library of clones. Thus, the term covers clones encoding a thiaminase or portion of a thiaminase which are isolated from other non-thiaminase clones.
A polypeptide thiamin-depleting agent can be encoded by a full-length nucleic acid sequence or portion of the full-length nucleic acid sequence. In preferred embodiments the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence encoding a naturally-occurring thiaminase, a nucleic acid sequence that hybridizes to such a nucleic acid sequence, or a functional derivative of either. The nucleic acid may be isolated from a natural source by cDNA cloning, use of PCR primers, subtractive hybridization, or other means standard to the art; the natural source may be any organism which naturally produces a thiaminase, specifically including those described in the Detailed Description below, and the nucleic acid may be synthesized by the triester or other method or by using an automated DNA synthesizer.
The term xe2x80x9chybridizexe2x80x9d refers to a method of interacting a nucleic acid sequence with a DNA or RNA molecule in solution or on a solid support, such as cellulose or nitrocellulose. If a nucleic acid sequence binds to the DNA or RNA molecule with high affinity, it is said to xe2x80x9chybridizexe2x80x9d to the DNA or RNA molecule. The strength of the interaction between the probing sequence and its target can be assessed by varying the stringency of the hybridization conditions. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Examples of hybridization conditions are shown in the examples below. Those skilled in the art will recognize how such conditions can be varied to vary specificity and selectivity. Under highly stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or two mismatches out of 20 contiguous nucleotides.
The invention also features recombinant nucleic acid encoding a thiamin-depleting agent, preferably in a vector effective to initiate transcription in a host cell. The vector may be in such a eukaryotic host cell or in vivo in cells of an organism. The recombinant nucleic acid can, for example, contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a thiamin-depleting agent polypeptide and a transcriptional termination region functional in a cell. While recombinant nucleic acid encoding an unmodified thiaminase, for example in a eukaryotic expression vector, from Bacillus thiaminolyticus can be utilized in the methods of this invention, in certain embodiments the encoded thiamin-depleting agent is different from that enzyme, and in other embodiments is not a modification or derivative of that thiaminase.
Thus, in a related aspect the invention provides a eukaryotic expression vector which includes a nucleic acid sequence encoding a thiamin-depleting agent. The expression vector is constructed and adapted for expression in eukaryotic cells, preferably in human cells. Preferably the vector does not include an origin of replication functional in eukaryotic cells. While vectors based on viral sequences can be beneficially used, in preferred embodiments, the vector is a non-viral vector, meaning that the vector does not contain sufficient viral sequences to cause viral replication or capsid formation. In certain embodiments, the encoded thiamin-depleting agent differs from a full-length thiaminase from Bacillus thiaminolyticus and in other embodiments is not a modification or derivative or that thiaminase. Other preferred embodiments are as described above for the nucleic acids and nucleic acid delivery methods.
In another related aspect, the invention provides a vector which includes a recombinant nucleic acid sequence which encodes a polypeptide thiamin-depleting agent, such as a thiaminase or a thiaminase derivative or thiamin-binding compound or derivative which is different from a Bacillus thiaminolyticus thiaminase. In preferred embodiments, the vector is an expression vector which is constructed and adapted for expression in prokaryotic cells, for example, E. coli, though a variety of other bacteria can be used. In other embodiments the vector is a eukaryotic expression vector, which is constructed and adapted for expression in eukaryotic cells. Other preferred embodiments are as described for the vectors, nucleic acids and nucleic acid delivery methods above.
In accord with the vectors and methods for delivery of nucleic acid encoding a thiamin-depleting agent, the invention also provides a eukaryotic cell transfected with a eukaryotic expression vector containing a nucleic acid sequence encoding a thiamin-depleting agent. Preferably, the cell is a vertebrate cell in vivo in a vertebrate organism, such as a bird or a mammal, e.g., a human. The thiamin-depleting agent can be any peptide or polypeptide compound, such as those described in the above aspects.
In another related aspect, the invention provides a composition for delivery of a nucleic acid sequence encoding a thiaminase or a thiaminase derivative to vertebrate cells in vivo. The composition includes a nucleic acid sequence encoding the thiaminase or a thiaminase derivative. The composition preferably also includes a component associated with a nucleic acid sequence which enhances delivery of the nucleic acid into the cells. In preferred embodiments, the nucleic acid and other components of the composition are as described above in connection with methods involving delivery of a nucleic acid sequence.
Thiamin-depleting agents, such as thiaminases and thiamin-binding compounds obtained from natural sources will be useful as described for the methods of this invention, and for analysis for constructing derivatives and synthetic thiamin-cleaving and thiamin-binding compounds. Thus, another aspect of the invention features an isolated, enriched, or purified polypeptide thiamin-depleting agent which has not previously been obtained. In the case of an agent which has been enriched or partially purified, the invention provides a purified agent, so that the agent is separated from at least 95%, preferably from at least 98%, and still more preferably from at least 99% of the macromolecules from the environment in which the agent is naturally produced. The agent therefore differs from a Bacillus thiaminolyticus thiaminase or mutated form of that thiaminase involving substitution or deletion of less than 1%, 5%, or 10% of the amino acid sequence of that thiaminase. In preferred embodiments, the agent is a thiaminase or thiaminase derivative or other polypeptide thiamin-cleaving compound. Also in preferred embodiments the agent is a thiamin-binding compound or derivative.
By xe2x80x9cpolypeptide thiamin-depleting agentxe2x80x9d it is meant an amino acid sequence which has antithiamin activity under conditions approximating human intracellular or extracellular conditions. In some cases the sequence is substantially similar to at least a portion of the amino acid sequence of a naturally occurring thiaminase or thiamin-binding compound. A sequence that is substantially similar will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence or portion of the sequence of the naturally occurring thiaminase or thiamin-binding compound. In other embodiments, the amino acid sequence has at least one change, such as a chemical modification of an amino acid or incorporation of at least one non-natural amino acid.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 6, 12, 18 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. In this invention, the polypeptide will commonly have at least about 50, 100, 200, or 400 amino acids conjugated together. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide it is meant that the specific amino acid sequence constitutes a significantly higher fraction (2- to 5-fold greater) of the total of polypeptide present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other polypeptides present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other polypeptides of about at least 2-fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no polypeptide from other sources. The other source polypeptide may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector. The term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired polypeptide.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g, in terms of mg/ml). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In another aspect the invention features an isolated, enriched, or purified polypeptide thiamin-depleting agent fragment, specifically including a thiaminase fragment. Preferably the polypeptide is a xe2x80x9crecombinant polypeptidexe2x80x9d.
By xe2x80x9ca polypeptide thiamin-depleting agent fragmentxe2x80x9d is meant an amino acid sequence that is less than a full-length sequence of a naturally occurring thiamin-depleting agent or equivalent length modification of such an agent, such as a shortened form of a thiaminase. Examples of such fragments include catalytically active fragments or mutant polypeptides, such as catalytically active thiaminase regions or mutants.
By a xe2x80x9cpolypeptide thiamin-depleting agent mutantxe2x80x9d is meant a polypeptide which differs from the native sequence of the corresponding polypeptide thiamin-depleting agent in that one or more amino acids have been changed, added or deleted. Changes in amino acids may be conservative or non-conservative. By xe2x80x9cconservativexe2x80x9d it is meant the substitution of an amino acid for one with similar properties such as charge, hydrophobicity, structure, etc. Examples of polypeptides encompassed by this term include, but are not limited to, (1) chimeric proteins which comprise a portion of a thiamin-depleting agent polypeptide sequence fused to a non-thiamin-depleting agent polypeptide sequence, for example an antibody or antibody fragment polypeptide sequence, and (2) thiamin-depleting agent proteins having a point mutation or a deletion. A thiamin-depleting agent mutant will retain some useful function such as, for example, binding to thiamin, or thiamin-cleaving catalytic activity. Thiamin-depleting agent mutants specifically include thiaminase mutants.
The term xe2x80x9crecombinant polypeptide thiamin-depleting agentxe2x80x9d is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. Of particular interest are recombinant thiaminases and recombinant thiaminase derivatives.
In accord with the above methods for inducing apoptosis and for treating neoplastic disorders, the invention also provides a pharmaceutical composition which includes at least one thiamin-depleting agent. Such a thiamin-depleting agent may be of any type, such as those pointed out above. Thus, in preferred embodiments, the thiamin-depleting agent is thiamin-cleaving compound such as a thiaminase or thiaminase derivative, a thiamin-binding compound or derivative, or a thiamin antagonist. Also, in preferred embodiments, the composition also includes a delivery targeting component. Such a component results in preferential delivery of the agent to a selected group of cells. Also in preferred embodiments, the composition includes a pharmaceutically acceptable carrier or excipient. Also in preferred embodiments, the composition includes a plurality of thiamin-depleting agents, and/or the composition includes an anti-neoplastic agent other than a thiamin-depleting agent. In these and other preferred embodiments, the thiamin-depleting agent, and other components of the pharmaceutical composition have characteristics as described above. Thus, preferred embodiments of such pharmaceutical compositions include compositions with components as described in other aspects herein or in the Detailed Description.
In a related aspect, the invention provides a method for making a pharmaceutical composition which includes a thiamin-depleting agent or a plurality of such agents. The method includes identifying a compound having thiamin-depleting activity, synthesizing the compound in an amount sufficient to produce a therapeutic effect by the induction of apoptosis of a selected group of cells, and preparing the compound in a pharmaceutically acceptable composition. Preferably the thiamin-depleting activity is thiamin-cleaving activity.
Similarly, the invention provides a method for making a pharmaceutical composition which includes a nucleic acid sequence encoding a thiamin-depleting agent. The method involves identifying a nucleic acid sequence encoding a peptide or polypeptide having thiamin-depleting activity, synthesizing the nucleic acid sequence in an amount sufficient to produce a therapeutic effect by the induction of apoptosis of a selected group of cells by expression from the nucleic acid sequence, and preparing the nucleic acid sequence in a pharmaceutically acceptable composition.
In preferred embodiments, the above aspects concerning methods of making, the composition includes components as described in other aspects for pharmaceutical compositions or compositions utilized in methods of inducing apoptosis, methods of killing cells, and methods of treating a disorder, or other methods of utilizing thiamin-depleting agents as described herein.
In this context, the term xe2x80x9csynthesizingxe2x80x9d refers to the artificial production of a molecule from one or more precursor molecules. Such production may involve chemical synthesis and/or may involve expression from a nucleic acid template. Such production may involve preparation of a molecule by combining two or more molecules, such nucleotides or amino acids, and/or may involve chemical modification of a single molecule, such as by the addition or removal of one or more substituent groups.
In view of the use of thiamin-depleting agents to induce apoptosis of vertebrate cells by reducing the level of thiamin, the invention also provides a method for identifying thiamin-depleting agents able to induce apoptosis of vertebrate cells. The method involves providing and contacting vertebrate cells with a compound having antithiamin activity. The induction of apoptosis of the cells indicates that the compound has the specified apoptosis inducing ability. Preferably, the cells are in contact with the anti-thiamin compound for a significant period. The appropriate time period can be selected based on the characteristics of the cells and the expected time needed to induce apoptbsis of the cells by thiamin-depletion. In preferred embodiments the time period is within a factor of about two of the time previously observed for the induction of apoptosis using Naegleria gruberi thiaminase in vitro, preferably a period of about 3-30 days, more preferably about 3-20 days, still more preferably about 3-10 days. An appropriate time period also can relate to the type of antithiamin activity which it is desired to detect. Thus, a longer period may be useful for detection of thiamin-binding compounds than thiamin-cleaving compounds. Those skilled in the art will recognize that the test period can be adjusted based on empirical evaluation of results obtained.
In the context of this invention xe2x80x9cantithiamin activityxe2x80x9d refers to a chemical or biological reaction or interaction which reduces the ability of a cell to utilize thiamin provided in the cellular environment. Thus, such an activity includes, for example, a thiamin-cleaving activity such as that of thiaminases. It also includes thiamin-binding activity such as that of previously identified thiamin-binding compounds or other thiamin sequestering effect. It further includes thiamin antagonist activity which can, for example, include competitive binding of the antagonist to thiamin carriers or enzymes which utilize thiamin as a substrate. The antithiamin activity can also include the inactivation or inhibition of an enzyme or other biomolecule which is necessary for the cellular incorporation of thiamin.
In preferred embodiments, the compound having antithiamin activity is a thiamin-cleaving compound, such as a thiaminase or thiaminase derivative, a thiamin-binding compound or derivative, or a thiamin antagonist.
In view of the useful treatment methods involving thiamin-depleting agents, the invention also provides a method of screening for such agents. The method involves contacting thiamin in solution with a plurality of test compounds and determining whether any of the compounds have antithiamin activity, where the antithiamin activity is a thiamin-cleaving activity or a thiamin-binding activity. Preferably, the compounds being tested are small molecules or are compounds selected on a chemical structural basis to be likely to have the antithiamin activity.
In another related aspect, the invention provides a method of screening compounds having antithiamin activity by contacting cells which do not synthesize thiamin but which require the presence of thiamin with one or preferably more test compounds and determining whether the presence of the test compound inhibits the cellular uptake or utilization of thiamin by the cell. The inhibition of cellular uptake or utilization of thiamin is indicative that the test compound is a thiamin antagonist. In preferred embodiments, the inhibition of uptake or utilization of thiamin is provided by an antithiamin activity as described above, such as thiamin-cleaving activity, thiarnin-binding activity, competitive inhibition, and inhibition of an enzyme required for thiamin utilization.
The invention also provides a method for screening synthetic compounds or derivatives of compounds to identify compounds having antithiamin activity. The method involves testing a plurality of compounds to determine whether the compounds have activity against thiamin in solution. In some embodiments in which derivatives of compounds having antithiamin activity are screened, the derivatives are not mutated forms of Bacillus thiaminolyticus thiaminase, or are not derivatives of that thiaminase. In preferred embodiments, the plurality of compounds are at least a portion of a compound library, such as a synthetic compound library or a combinatorial library, or derivatives of thiaminases or thiamin-binding compounds. Also in preferred embodiments, the antithiamin activity being determined is thiamin-cleaving activity or thiamin-binding activity.
The term xe2x80x9cmethod of screeningxe2x80x9d refers to a method for evaluating a plurality of test compounds to determine whether one or more test compounds possess a particular functional property and may also determine the level of activity associated with that functional property, but is distinct from a method for merely evaluating the level of activity of a compound which is known to have a particular activity. The method of screening is suitable for and is used to evaluate a plurality, preferably a large number of test compounds, e.g., at least 10, more preferably at least 100 and still more preferably at least 1000 test compounds.
In an alternative to the use of thiamin depleting agents for the therapeutic induction of apoptosis, a treatment can be utilized which acts on at least one critical step or component in the cellular apoptosis pathway that is activated as a consequence of thiamin depletion. Thus, for example, the treatment can modulate, usually inhibit, a step in a crucial metabolic pathway, or the transmission of a signal through a signaling receptor. Preferably, the treatment involves the localized administration or activation of a compound, leading to apoptosis. The targeting or localization may be accomplished by a variety of means, including those described herein, selected as appropriate for the type of treatment or molecule to be administered.
In the context of the apoptosis induction pathway associated with thiamin deficiency, the term xe2x80x9ccriticalxe2x80x9d means that appropriate modulation of the step or activity of a component or reaction results in the induction of apoptosis. This does not require, though it is preferable, that the step be essential to all pathways in the induction of apoptosis. For example, alternate reactions to an inhibited reaction may also induce apoptosis, but even if there are multiple pathways to apoptosis, all that is needed is that one crucial step or activity be modulated such that apoptotic cell death is triggered.
Still further, this invention provides additional methods for inducing apoptosis of a selected group of cells by creating a localized deficiency of a nutritional factor different than thiamin. Deficiencies of a number of different nutritional factors in addition to thiamin induce apoptosis. It has, for example, been demonstrated that depletion of iron or glucose induces apoptosis of cells. Thus, depletion of such a nutritional factor in the targeted group of cells can be used as described herein for thiamin deficiencies. Such targeted nutritional deficiencies can be used in place of or in addition to a localized thiamin deficiency. In addition, deficiencies of certain other nutritional factors do not induce apoptosis, but do induce quiescence. An example of such a quiescence-inducing factor is isoleucine. Factors which induce quiescence are thus not useful for directly inducing apoptosis, but could be used in conjunction with a thiamin deficiency or other apoptosis-inducing nutritional factor deficiency (or even other other apoptosis-inducing methods) in cases where the apoptosis induction only or predominantly takes place with quiescent cells rather than actively growing cells in order to increase the proportion of quiescent cells among the targeted cells.
In preferred embodiments, such methods include the targeting methods or agents or other composition components, or accessory methods as described in connection with the use of LAIDT above.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.